The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase

Systemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z score of [~]0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited inhibitory effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1{Delta}{Delta} mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a Ki of 1,550 {+/-} 245.6 nM. Thus, this compound has the potential for development into an antifungal compound.